b On day 3 postburn, epidermic cells pro-gressed to vacuolation, collagenous fibers in superficial dermis had hyaline degeneration and scattered inflammatory cells infiltrated in dermis
Trang 1Fig 37 a Appearance of deep second-degree burns wound on day 1
postburn HE !100 b On day 3 postburn, epidermic cells
pro-gressed to vacuolation, collagenous fibers in superficial dermis had hyaline degeneration and scattered inflammatory cells infiltrated in
dermis HE !100 c On day 5 postburn, superficial tissues in dermis
were liquefactively necrotic and exfoliated Inflammatory cells, mainly neutrophil and occasionally with few macrophages,
infil-trated HE !100 d On day 10 postburn, the appearance of epithelia islands was seen HE !100 e On day 10 postburn, sweat glands in
dermis proliferated with the infiltration of inflammatory cells all round, mainly neutrophils, as well as many lymphocytes and
mono-nuclear macrophages HE !100 f On day 15 postburn, the adnexal
epithelia in deep dermis grew vertically from the deep, then migrated
toward and covered the wounds HE !100 g Most wounds were
covered with stratified squamous epithelium Actively proliferated fibroblasts and neoformative capillaries were visible in the dermis,
accompanied by the infiltration of lymphocytes HE !100 h
Neo-formative skin has an approximately normal structure HE !100.
when the necrotic epidermis exfoliated, superficial tissue
in the dermis liquefied and loosened due to necrosis and
neutrophilic infiltration (occasional lymphocyte and
mononuclear macrophages) (fig 37c) On day 7
postin-jury, the aforementioned pathological changes became
more notable On day 10, the superficial necrotic tissue in the dermis liquefied, discharged and thinned A large amount of neutrophils infiltrated into the liquefied ne-crotic tissue, interwoven with few mononuclear macro-phages The dermal adnexal epithelia in the underlying
Trang 2deep tissue revealed squamous metaplasia with enlarged
cells, deep karyon, rich and red cytoplasm that clustered
to form ‘epithelium islands’ (fig 37d) Among these
is-lands, fibroblasts proliferated and were characterized by
large volume of cells, conspicuous nucleolus (binucleolate
was frequently present) and basophilic cytoplasm In
ad-dition, infiltration of many inflammatory cells including
neutrophils (mainly), lymphocytes and mononuclear
macrophages was visible in wounds (fig 37e) On days 15
and 20 postburn, an inflammatory exudation layer
re-placed the necrotic layer whose liquefaction was now
accomplished The underlying residual adnexal epithelia,
fibroblasts and endothelia showed active proliferation
The regenerated ‘epithelial islands’ grew vertically, then
migrated toward and covered the wounds (fig 37f, g)
Neoformative capillaries were noticed and granulation
tissues were formed The infiltration of inflammatory
cells (mainly lymphocytes) in the dermis was still present,
especially in the periphery of the regenerated skin
ap-pendages After healing, regenerated skin appeared to be
almost normal in structure and the majority of skin
appendages were restored completely Infiltration of
cer-tain inflammatory cells and few macrophages in dermis
persisted (fig 37h)
Electron-Microscopic Observation
Ultrastructural alterations of the superficial dermis
layer were observed on days 1, 3 and 5 postburn, while
those of full-thickness skin were done on days 7, 10, 15
and 20 postburn and after healing
On day 1 postburn, electron-microscopic observation
indicated loose and disorderly arranged collagenous fibers
in the superficial dermis, and a distinct and recognizable
light-dark zone of the fasciculus Fibroblasts which had a
cytomembrane profile which remained the same although
being deprived of the cell organ Nuclear membrane
loomed with increscent nucleopore to cause the
interpola-tion of nuclein and chromatin, leading to lumpish
chro-matin Microsangial endothelia had similar changes as
did the fibroblasts The vascular walls appeared as a
lamellar membranous structure In the lumen, we noted
hemagglutination, frequently joined with blood platelet
adherence to the vascular walls (fig 38a) On day 3
post-burn, collagenous fiber bundles in the superficial dermis
varied in thickness and appeared disorderly with faint or
absent light-dark zones The cell ultrastructure
interwo-ven between fibers disappeared On day 5 postburn, the
superficial collagenous fibers merged to produce floccules
with high electron density surrounded by scattered
elec-tron-dense organelle-like structures and necrotic cell
frag-ments as well as occasionally by varied lower
electron-dense lipid droplets without coating of the limiting
mem-brane (fig 38b) On day 7, when the ultrastructural
change of the superficial dermis was similar to that on
38
day 5, deep collagenous fibers and the structure of dermal adnexal epithelia nearly returned to normal, except for a slight broadening of the intercellular space On day 10 postburn, fibroblasts in the dermis showed condensed nuclear chromatin, hypertrophic and shift aside nucleoli Rough endoplasmic reticulum (RER) proliferated show-ing flat vesicular and vesicular-like expansion, while re-sidual dermal adnexal epithelia were distributed in clus-ters containing a high amount of tonofibrils (fig 38c) On days 15 and 20 postburn, collagenous fiber bundles of der-mis varied in thickness and appeared disorderly; fibro-blasts were binucleated and showed obvious nucleoli with the presence of karyosome; perinuclear pool demon-strated beading swelling and the RER proliferated (fig 38d) In dermal adnexal epithelia, nucleoli were dis-tinct and increased numbers of karyosomes were fre-quently observed
The chondriosome was rich and intracytoplasmic to-nofibril showed an increase in cytoplasmic presence Also,
we observed a gradual establishment and improvement of the intercellular desmosomal junction After healing,
Trang 3neo-Fig 38 a Vascular wall appeared in the lamellar membranous
struc-ture In the lumen, there was hemagglutination, frequently joined
with blood platelet adhering to vascular walls TEM !6,000 b
Col-lagenous fibers fused each other, occasionally with cell collapse.
TEM !6,000 c Dermal adnexal epithelia distributed in clusters,
with near-membrane nucleolus and expanded endoplasmic
reticu-lum Many tonofibrils are contained in the cytoplasm TEM.
!6,000 d On day 15 postburn, fibroblasts showed active protein
synthesis and metabolism TEM !6,000 e After wound healing, the
entire dermis-epidermis junction can be seen TEM !8,000 f
Fi-broblasts in dermis showed normal and collagenous fibers were
uni-form in thickness and arranged in an orderly fashion (after healing).
TEM !10,000 g The healing skin after deep second-degree burns is
almost the same as noninjured skin.
formative epidermis cells and intercellular junction
re-covered almost to normal and the integrated
dermis-epi-dermis junction reappeared (fig 38e) The majority
fibro-blasts in the dermis appeared as a long strip rich in
cyto-plasm with well-developed organelles However, no
addi-tional active metabolism of the same was observed Fibro-blasts were uniform in thickness, fasciculated and well orientated (fig 38f) The healing skin of deep second-degree wounds became almost identical to that of nonin-jured skin (fig 38g)
Trang 4Pathological changes of the deep second-degree burn
wounds treated with BRT with MEBT/MEBO can be
divided into three stages: (1) denaturation and necrosis,
(2) liquefaction, and (3) restoration These stages may
overlap Burns wounds healed by complete physiological
regeneration
Discussion
Among the available literature regarding the
pathologi-cal changes of burns wounds treated with BRT with
MEBT/MEBO, the majority report the light microscopic
observation before and after treatment with MEBT [2–4]
There are relatively few monographs or systemic study
reports on the ultrapathologic processes during BRT with
MEBT/MEBO
Our study demonstrated that the pathological changes
of burns wounds treated with BRT with MEBT/MEBO
were totally different from pathological processes when
treated conventionally [5, 6] On days 1–3 postburn, the
so-called ‘acute inflammatory response period’ with its
typical denaturation and necrosis occurred in the burned
epidermis and superficial dermis, revealing a slight
in-flammatory response, but without signs of a ‘leukocyte
infiltration zone’ Then the necrotic tissue began to
lique-fy and discharge increasingly as the disease course
pro-gressed Such changes climaxed around day 10 when
der-mal adnexal epithelial cells, fibroblasts as well as other
repaired cells showed signs of regeneration Microscopic
observation indicated large cell bodies of fibroblasts,
increased basophil of the cytoplasm, hypertrophic
nu-cleoli, and RER which proliferated, demonstrating the
typical flat vesicular and vesicular-like expansion At this
time, the proliferation and migration of residual dermal
adnexal epithelia and proliferation of granulation tissues
became dominant The most spectacular observation was
that the ‘epithelia island’ (formed by the regenerated
adnexal epithelia) initially grew vertically, then migrated
toward and covered the wound as granulation tissues
pro-liferated This resulted in a flat skin surface During this
period, either residual adnexal epithelia or fibroblasts
dis-played ‘active protein synthesis and metabolism’ [7] The
prominent characteristic changes involved significant
proliferation of RER and the appearance of increased
karyosomes Finally, the wounds were noted to be
com-pletely covered by regenerated squamous epithelia as
healing was accomplished
We observed that inflammatory cells demonstrated a
series of responses during wound healing On day 1
post-burn, we observed infiltration of neutrophils into the
der-mis followed by lymphocytes and mononuclear
macro-phages This infiltration increased on day 3 Neutrophils
were the predominant infiltrating cells in necrotic tissue,
and their numbers grew or declined depending upon the length of liquefaction time Meanwhile, lymphocytes and mononuclear macrophages, mainly located in the residual dermis, dramatically increased during the period of time that necrotic tissue was being rejected and wound repair was initiated These cells remained the dominant cells impacting wound repair subsequent to the liquefaction period This study does not resolve questions as regards the significance of the space-time distribution of these cells nor does it answer definitively questions about sequential changes These questions merit further study However, we do note the remarkable truth that, during wound healing, the function and activity of the above cells directly or indirectly, alone or cooperatively, participated
in and regulated wound healing
The study results indicated a staged pathological change of burns wounds when treated with BRT with MEBT/MEBO Wounds changed in three stages accord-ing to the different time phase postburn: (1) denaturation and necrosis stage, (2) liquefaction stage, and (3) stage of repair by regeneration, which may overlap during wound healing
The first stage began subsequent to burn injury till day
3 and the pathomorphological change was characterized
by the denaturation and necrosis of burns tissue from which we derived the name ‘denaturation and necrosis stage’ We postulated that these changes were the result of direct thermal exposure, local microcirculation blockage and other secondary injuries The minor inflammatory response at this stage had a close correlation with the effects of MEBT/MEBO On day 5 postburn, wound liquefaction became conspicuous and climaxed on day
10 After this time, liquefaction diminished and we noted less necrotic tissue The inflammatory response, however, remained correlated with the liquefaction of necrotic tis-sue Repair by regeneration was noted on day 10 postburn and was manifested mainly by active proliferation of residual dermal adnexal epithelia This stimulated the proliferation of peripheral fibroblasts and endothelia, and further formed granulation tissues, leading to final heal-ing by epithelization After experiencheal-ing further differen-tiation and reformation, the regenerated skin finally at-tained the structure of normal skin This study demon-strates that the effect of BRT with MEBT/MEBO deliv-ered similar pathological changes for both deep second-degree and third-second-degree burns wounds The only differ-ence lay in the ultimate healing modes
Remarkably, this study revealed that the healed wounds appeared as flat fully regenerated skin, featuring restored, viable hair, almost normal skin elasticity and
no scar formation Histomorphological observation con-firmed that the epidermal cells of neoformative skin had the equivalent structure as normal epidermis and that the epidermis had good joints with dermis papilla The healed
Trang 5skin demonstrated a fully functional and integrated
der-mis-epidermis junction The initially infantile and active
fibroblasts gradually grew into stable fibrocytes
Arterio-lae and venuArterio-lae with thick walls and integrated structure
replaced the neoformative capillaries Collagenous fibers
were uniform in thickness and orientated fascicularly,
without the presence of whirl-like and nodular
arrange-ment Follicles, sweat glands and sebaceous glands as well
as other dermal accessory organs also regenerated
com-pletely Therefore, we concluded that treatment of deep
second-degree burns wounds with BRT with MEBT/
MEBO results in complete physiological regeneration
with minimal scar formation
References
1 Xu RX: Brief introduction on Chinese moist medical science for burns.
Chin J Burns Wounds Surface Ulcers 1996;8:1–6.
2 Wang GS, Jian WG: Pathological changes of rabbit with experimental
burns treated by MEBO and investigation of the mechanism of action.
Chin J Burns Wounds Surface Ulcers 1992;4:7–11.
3 Zhang XZ, Cheng SR: A case report: Electron microscopic observation on
burn wound treated with MEBO Chin J Burns Wounds Surface Ulcers
1992;4:6–9.
4 Zhao YL, Wang Y: Electron microscopic observation on the effect of
MEBO in treating deep burn wound Chin J Burns Wounds Surface Ulcers
1995;7:1–5.
5 Fang ZY, Wu ZL: Burn Theory and Practice Shenyang, Liaoning Science
and Technology Publishing House, 1989, pp 12–14.
6 Fu XB, Wang DW: Basis of Trauma Repairing Beijing, People’s Military
Medical Press, 1997, pp 14–30.
7 Wu ZB: Basis of Ultra-Micro-Pathology Beijing, People’s Health Press,
1990.
Clinical Procedure and Histological Observation
of Full-Thickness Burns Treated with BRT with
MEBT/MEBO: A Case Report
Introduction
A 20-year-old female patient sustained a 35% TBSA
burn secondary to exposure to gasoline fire (15% deep
partial-thickness and 20% full-thickness loss) The patient
was hospitalized at 12 h postburn and was diagnosed as
suffering with full-thickness loss (third-degree) burns on
both lower extremities The epidermis was necrotic and
detached and the dermal layer was degenerated and
necrotic with a waxy yellow and waxy white appearance
(fig 39a) The pathological section examination of the
sampled local wound tissues revealed necrosis of
full-thickness epidermis and dermis, degeneration and
struc-tural disturbance of collagenous fibers in dermis, and
microcirculation stasis (fig 39b)
Results
After admission, the patient was treated initially with MEBO burns ointment to protect burn tissue before we performed skin cultivation and relief as per our BRT pro-tocol At 48 h postburn, the wounds began to liquefy and the liquefaction was complete by day 4 (fig 40) The liquefied products were gently removed from the wound surface before MEBO was reapplied every 3–4 h
Repeat biopsy at the same burned location was per-formed for pathological examination The results showed massive granular tissues among the necrotic epithelial tis-sue, a proliferation of newly regenerated epithelial cells with collagenous fibers, as well as the typical skin em-bryonic base (EB) (fig 41a, b) After a 10-day application
of MEBO, the comparably primitive epithelial tissues were observed under pathological examination of epithe-lial tissues sampled from the wound edge
Fig 39 a A 20-year-old female patient sustained a 35% TBSA burn
secondary to exposure to gasoline fire (15% deep partial-thickness
and 20% full-thickness loss) b Necrosis of full-thickness epidermis
and dermis, degeneration and structural disturbance of collagenous fibers in dermis, and microcirculation stasis HE !20.
Trang 6Fig 40 On day 4, the wounds began to liquefy under the action of
MEBO, and the wound skin began to regenerate.
At 20 days post-treatment with MEBO, the pathologi-cal examination of deep burns wounds tissue showed the presence of the newly formed intact stratified squamous epithelium The epithelial cells of the superficial layer appeared normal Appearance of the collencytes and mi-croangium in the dermis layer was typical On day 30 post-treatment, epithelial tissues showed a remarkable degree of regeneration (fig 42a), and skin structure was almost normal (fig 42b)
Immunohistochemical Examination
Twenty days post-treatment with BRT with MEBT/ MEBO, wound tissue was examined and the results showed the clear appearance of collagenous fibers in epi-thelial tissue and subcutaneous tissue Argentaffin stain
Fig 41 a Group proliferation of newly regenerated epithelial tissues
and collagenous fibers as well as typical skin embryonic base (EB).
b The skin EB formed by microangium, collagenous fibers and
epi-thelium (stem cell) in newly regenerated epithelial tissues HE !40.
Fig 42 a On day 30 post-treatment, wound tissue showed good
pro-cess of regeneration b Day 30 post-treatment, epidermis and dermis
showed physiological structure.
Trang 7indicated active regeneration of reticular fibers The
re-ticular fibers around the small blood vessels of
subcuta-neous tissue appeared relatively normal The basal cells of
epidermis regenerated actively (fig 43a, b)
Thirty days after BRT with MEBT/MEBO treatment,
the regenerated and repaired epithelial tissue was AE3
stained The pictures showed positive proteins of
squa-mous epithelium, brown-stained cytoplasm and
blue-stained nucleus of granular cells in epidermis (fig 43c, d)
AE1 stain showed negative proteins of glandular epi-thelium, which is representative of glandular epithelium already transformed into squamous epithelium (fig 43e)
Electron-Microscopic Observation
On the day of injury, the epithelium showed necrotic degeneration, and the monocytes showed a clear nuclear shift with karyopyknosis and even phagocytosis (fig 44) Five days post-treatment with MEBO, we noted active growth of fibrocytes and fibroblasts (fig 45)
Fig 43 a Active regeneration of the basement membrane in the basal
lamina of the epidermis Argentaffin !40 b Reticular fibers around
the small blood vessel in the dermis stained black Argentaffin !40.
c Positive protein of squamous epithelium (indicating spontaneous
self-regeneration and repair) AE3 !20 d Same as figure 46a AE3.
!40 e Negative protein of glandular epithelium AE1 !20.
Trang 8Fig 44 On the day of injury, nuclear shift, karyopyknosis and
phago-cytosis of monocyte is seen TEM !8,000.
Fig 45 Five days post-treatment with BRT, active growth of
fibro-blast TEM !2,000.
Ten days post-treatment with BRT with MEBT/
MEBO, we noted the appearance in epidermis of
echino-cytes and desmosome in the stratum spinosum (fig 46a,
b), granular cells in stratum granulosum, and
melano-somes in the basal cell layer (fig 46c, d) We also observed
phagocytes active around the small vessels, indicating
recovery of function (fig 46e)
Twenty days post-treatment, we noted the appearance
of the hemidesmosome junction between the basal cell
layer and epithelia Active mitochondria and RER in
fibroblasts also appeared (fig 47a–c)
Thirty days post-treatment with BRT with MEBT/
MEBO, with the regeneration and repairing of epithelium
almost complete, collagenous fibers were mature with a
diameter of 0.1–0.5 Ìm and arranged in an orderly fashion (fig 48) Light and dark periodic cross-striation (64 nm) was also observed No pathomorphological changes of collagenous fibers such as distortion, helicoid (whirlpool) or cauliflower-like were observed
After wound healing, functional exercises and physio-therapy of the lower extremities were required MEBO was continued as ordinary skin oil The patient healed and was discharged home on day 45 postburn
Effect of BRT with MEBT/MEBO on the Expression and Regeneration of Epidermal Regenerative Stem Cells
Introduction
As an innovative therapeutic system in burns therapy, BRT with MEBT/MEBO has enjoyed wide clinical accep-tance as part of a protocol including the topical drug oint-ment MEBO This therapeutic system successfully solved four major clinical problems: pain, wound infection, pro-gressive necrosis, and scarring in deep second-degree burns wounds Recently, a new landmark innovation, the regeneration and replication of skin tissue in the subcuta-neous fat tissue of full-thickness burns wounds, has been accomplished by this innovative protocol We know that
no stem cells remain in the basal layer of epidermis of deep second-degree and superficial third-degree burns wounds Therefore, we investigated the source of the regenerative epidermal stem cells which makes the fatty layer burns wounds repair spontaneously This study was designed to observe dynamic changes in the regenerative epidermal stem cells of deep burns wounds tissues using the immunocytochemistry method
Materials and Methods
Tissue samples were taken from both normal skin and burns wounds of the following 2 burns patients who received BRT with MEBT/MEBO treatment as first aid immediately after the burns incident.
Case 1 A 6-year-old boy was scalded by hot water on his back and
both lower limbs with an area of 33% TBSA and depth of deep sec-ond-degree.
Case 2 A 24-year-old male sustained flame burns on his four
extremities with an area of 25% TBSA, and depth of deep second-degree and superficial third-second-degree.
Tissue samples of the injured areas were taken from wounds of the 2 patients at 24 h and on 4, 7, 14, 21 and 28 days postburn The samples were placed in plastic tubes and frozen immediately in liq-uid nitrogen, then embedded in Tissue-Tek OCT Compound and frozen in liquid nitrogen Sections of 10 Ìm thickness were made in a constant freezing microtome.
Trang 9Fig 46 a 10 days post-treatment Desmosome junctions among four
echinocytes TEM !3,500 b Desmosome 10 days post-treatment with MEBO TEM !20,000 c Granular cells 10 days post-treat-ment with BRT with MEBT/MEBO TEM !20,000 d
Melano-somes in basal cell layer 10 days post-treatment with BRT with
MEBT/MEBO TEM !6,000 e 10 days post-treatment, phagocytes
show active phagocytosis TEM !6,000.
Indirect immunofluorescence staining with a biotin-avidin DCS
system was performed The frozen section was incubated with 10%
horse serum at 4° C for 20 min, then a diluted (1:20) solution of
mouse anti-human keratin type 19 monoclonal antibody (the 1st
antibody) was added and the mix was again incubated overnight at
4° C Subsequent to washing with phosphate buffer solution, the
sec-tion was added to 7.5 Ìg/ml of biotinized horse mouse IgG
anti-body (the 2nd antianti-body, Vector Laboratories, Burlingame, Calif.,
USA) and incubated at 4° C for 1 h After another washing, 10 Ìg/ml
of biotin-avidin DCS (Vector Laboratories) was added and incubated
at 4° C for 1 h The section was rinsed and then mounted with
glycer-in contaglycer-inglycer-ing 10% PBS and 1% p-phenylenediamglycer-ine A control
sec-tion of normal skin was stained in the same way, but without adding the 1st antibody All specimens were observed under an Olympus reflecting fluorescence microscope (Japan) and photos were taken using ASA400 KODAK films.
Trang 10Fig 47 a 20 days post-treatment, the epithelia adhered with
hemi-desmosome and fibroblast in the basal cell layer TEM !3,000 b 20
days post-treatment, active mitochondrion and rough endoplasmic
reticulum in the fibroblast TEM !17,000 c Same as figure 47b.
TEM !20,000.
Fig 48 30 days post-treatment with BRT with MEBT/MEBO, the
collagenous fibers are in orderly arrangement with a diameter of 0.1– 0.5 Ìm and have light and dark periodic cross-striation (64 nm) TEM !20,000.
Results
Immunocytochemical examinations were made on normal skin and burns wounds tissue sections treated with specific mouse anti-human keratin type 19 mono-clonal antibody The results revealed that in the normal controls of both cases, there were few positive numbers of epidermal stem cells with keratin type 19 (fig 49a) Wound tissue at 24 h postburn showed a moderate amount of positive epidermal regenerative stem cells (fig 49b) and on day 4 postburn, the number of positive epidermal stem cells around the sweat gland, capillaries and hair follicles increased (fig 49c) On days 7 (fig 49d) and 14 (fig 49e), epidermal stem cells containing human keratin type 19 continually increased and exceeded the level attained at day 4 postburn, before gradually reaching
a peak level Prior to days 21 (fig 49f) and 28 (fig 49g) postburn, the number of positive regenerative stem cells decreased to a certain level as the burns wounds pro-gressed to healing The observation showed that, after treatment with BRT with MEBT/MEBO, the prolifera-tion status of the potential regenerative stem cell of the burns patients changed at a regular rate We propose that regenerative stem cells may be the source of epidermal regenerative stem cells The glowing fluorescent cells found observed under the microscope represented the potential regenerative stem cells in the wound tissues of deep second- and superficial third-degree burns After treatment with MEBO, these stem cells may aid the deep partial thickness burns wounds to heal without scar for-mation and aid the superficial full-thickness burns wound
to regenerate skin while healing spontaneously